The recognition of the proteolytic substrates by the proteasome is arguably the most important step in the ubiquitin-proteasome pathway, and certainly the least understood. Highly purified proteasomes, composed of a barrel-shaped core particle (CP) bearing the sequestered protease active sites and a 17-subunit regulatory particle (RP), are competent to degrade ubiquitinated proteins. Because of this, it is generally assumed that the primary ubiquitin receptors for proteasomal degradation are subunits of the proteasome. Furthermore, since the CP alone is not competent to degrade ubiquitinated substrates, the ubiquitin receptor is thought to reside in the RP. While one subunit of the RP has been shown to bind ubiquitin chains, yeast cells deleted for this subunit are viable, indicating that there must be other functionally essential ubiquitin receptors. In the proposed work, binding of ubiquitin chains to the intact proteasome will be directly demonstrated for the first time using surface plasmon resonance. The RP has recently been fragmented into base and lid subcomplexes, and these subcomplexes will be tested for ubiquitin chain binding (Aim 1). By purifying lid and base complexes from other RP mutant strains, the lid or the base (whichever is relevant) will be further subdivided. In this way, the minimal RP subassembly competent for binding multiubiquitin chains will be defined (Aim 2). Genes encoding these candidate ubiquitin receptors will then be subjected to mutagenic analysis in order to identify the ubiquitin receptor (Aim 3). Mutant alleles of ubiquitin are known that are competent for the formation of ubiquitin conjugates on substrates, but that do not in turn target those substrates for degradation. This suggests that chains composed of mutant ubiquitin are not recognized by the ubiquitin receptor. Furthermore, at least one of these mutant ubiquitin alleles is lethal in yeast. Candidate ubiquitin receptors will be mutated and screened for allele-specific suppression of the lethality of mutant ubiquitin. Finally, the biochemical effect of the suppressor mutations will be confirmed by purifying intact proteasomes from the suppressor mutants and testing for the binding of mutant and wild-type ubiquitin chains.